中国农业科学2008,41(10):3249-3255ScientiaAgriculturaSinicadoi:10.3864/j.issn.0578-1752.2008.10.043收稿日期:2007-08-27;接受日期:2007-10-30基金项目:国家科技部奶业支撑计划(2006BAD04A12,2006BAD04A01)作者简介:狄和双(1979-),女,河北南皮人,博士研究生,研究方向为动物生殖生理与调控。Tel:15896006815;E-mail:dhsh1979@163.com。通讯作者王根林(1957-),男,江苏靖江人,教授,研究方向为动物生殖生理与调控。Tel:025-84395045;E-mail:glwang@njau.edu.cnTGF-β1对奶牛乳腺上皮细胞增殖、凋亡及HSP70蛋白表达的影响狄和双,杜娟,张中霞,黄静燕,崔群维,程凯宁,王根林(南京农业大学动物科技学院,南京210095)摘要:【目的】探讨TGF-β1对奶牛乳腺上皮细胞增殖、凋亡和HSP70蛋白表达的影响。【方法】TGF-β1作用乳腺上皮细胞不同时间,收集细胞及培养液,应用MTT检测细胞增殖、流式细胞术检测细胞凋亡率、比色法检测LDH活性、琼脂糖凝胶电泳检测DNA完整性、Westernbolt技术分析HSP70蛋白的表达量。【结果】1、5、10ng·ml-1的TGF-β1均能抑制乳腺上皮细胞的增殖,且随浓度增加抑制作用加大,呈现剂量依赖性,其中10ng·ml-1TGF-β1组与对照组差异显著(P<0.05);10ng·ml-1TGF-β1作用于乳腺上皮细胞,随着作用时间的延长,细胞凋亡率逐渐升高,其中6、12和24h与对照组相比差异极显著(P<0.01);LDH活力随作用时间延长而升高,12、24h与对照组差异极显著(P<0.01);DNA完整性随TGF-β1作用时间的延长发生不同程度降解;TGF-β1作用2~6h细胞大量表达HSP70,6h达到高峰,而后下降,2~12h的表达量均极显著高于对照组(P<0.01)。【结论】TGF-β1可以抑制奶牛乳腺上皮细胞的增殖,具有浓度依赖性。10ng·ml-1TGF-β1可以显著增加细胞凋亡率,提高细胞培养液中LDH活力,使细胞DNA发生不同程度的降解,同时HSP70蛋白随TGF-β1作用时间发生显著的表达变化。本试验结果表明,TGF-β1可以以促进上皮细胞凋亡的方式,促进奶牛乳腺发生退化。关键词:TGF-β1;乳腺上皮细胞;细胞凋亡;HSP70EffectsofTGF-β1onProliferation,ApoptosisandHSP70ExpressionofMammaryEpithelialCellsofDairyCowDIHe-shuang,DUJuan,ZHANGZhong-xia,HUANGJing-yan,CUIQun-wei,CHENGKai-ning,WANGGen-lin(AnimalScienceandTechnologyCollegeofNanjingAgriculturalUniversity,Nanjing210095)Abstract:【Objective】TheaimsofpresentstudyweretoinvestigatetheeffectsofTGF-β1onproliferation,apoptosisandHSP70expressionofthemammaryepithelialcellsofdairycow.【Method】BovinemammaryepithelialcellswereculturedinDMEM/F12+10%CSmediumwithTGF-β1andcellsandsupernatantwerecollected.Thecellproliferationandapoptosis,activityofLDH,integrityofDNA,andexpressionofheatshockprotein70(HSP70)weredetectedbyMTT,Flowcytometry,lactatedehydrogenase(LDH)kit,agarosegelelectrophoresisandWesternbolt,respectively.【Result】TheresultsshowedthatcellsproliferationswereinhibitedaftertreatedwithTGF-β1indosedependentway.TherateofapoptosiscellssignificantlyincreasedaftertreatmentwithTGF-β1for6,12,24hours(P<0.01).TheactivityofLDHwassignificantlyhigherinthetreatedgroupsthanthatinthecontrolgroup(P<0.01).ThenucleotideofDNAwasdegradatedgraduallyastreatmenttimeincreased.TheexpressionofHSP70increasedafterTGF-β1treatmentformorethan2hoursanditreachedthehighestlevelwhentreatedfor6h,thendecreased.【Conculsion】TGF-β1couldinhibittheproliferationofbovinemammaryepithelialcellsinadose-dependentway.Atadoseof10ng·ml-1,TGF-β1couldsignificantlystimulatecellapoptosisandincreaseLDHactivity,whichwereresultedindegradationofDNA.3250中国农业科学41卷Long-termcultureofbovinemammaryepithelialcellswithTGF-β1couldleadtotheup-regulationoftheexpressionofHSP70.ItissuggestedthatTGF-β1couldpromotetheapopt...